Journal of Environmental Science and Health, Part B Pesticides, Food Contaminants, and Agricultural Wastes
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Implication of zinc excess on soil health Jadwiga Wyszkowska, Edyta Boros-Lajszner, Agata Borowik, Małgorzata Baćmaga, Jan Kucharski & Monika Tomkiel To cite this article: Jadwiga Wyszkowska, Edyta Boros-Lajszner, Agata Borowik, Małgorzata Baćmaga, Jan Kucharski & Monika Tomkiel (2016) Implication of zinc excess on soil health, Journal of Environmental Science and Health, Part B, 51:5, 261-270, DOI: 10.1080/10934529.2015.1128726 To link to this article: http://dx.doi.org/10.1080/10934529.2015.1128726
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Date: 24 February 2016, At: 08:47
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 2016, VOL. 51, NO. 5, 261–270 http://dx.doi.org/10.1080/10934529.2015.1128726
Implication of zinc excess on soil health Jadwiga Wyszkowska, Edyta Boros-Lajszner, Agata Borowik, Ma»gorzata Bacmaga, Jan Kucharski, and Monika Tomkiel
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Department of Microbiology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
ABSTRACT
ARTICLE HISTORY
This study was undertaken to evaluate zinc’s influence on the resistance of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase and urease. The experiment was conducted in a greenhouse of the University of Warmia and Mazury (UWM) in Olsztyn, Poland. Plastic pots were filled with 3 kg of sandy loam with pHKCl – 7.0 each. The experimental variables were: zinc applied to soil at six doses: 100, 300, 600, 1,200, 2,400 and 4,800 mg of Zn2C kg¡1 in the form of ZnCl2 (zinc chloride), and species of plant: oat (Avena sativa L.) cv. Chwat and white mustard (Sinapis alba) cv. Rota. Soil without the addition of zinc served as the control. During the growing season, soil samples were subjected to microbiological analyses on experimental days 25 and 50 to determine the abundance of organotrophic bacteria, actinomyces and fungi, and the activity of dehydrogenases, catalase and urease, which provided a basis for determining the soil resistance index (RS). The physicochemical properties of soil were determined after harvest. The results of this study indicate that excessive concentrations of zinc have an adverse impact on microbial growth and the activity of soil enzymes. The resistance of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase and urease decreased with an increase in the degree of soil contamination with zinc. Dehydrogenases were most sensitive and urease was least sensitive to soil contamination with zinc. Zinc also exerted an adverse influence on the physicochemical properties of soil and plant development. The growth of oat and white mustard plants was almost completely inhibited in response to the highest zinc doses of 2,400 and 4,800 mg Zn2C kg¡1.
Received 22 August 2015 KEYWORDS
Enzymes; microorganisms; resistance index; soil; zinc
Introduction Environmental richness is one of the key determinants of biodiversity. Countless living organisms reside primarily in soil which hosts various biological and biochemical processes and acts as the ideal intermediary between the edaphon and surface vegetation. Rapid changes that contribute to environmental degradation can decrease the species diversity of living organisms.[1] A serious threat is posed by heavy metals which penetrate soil ecosystems, accumulate in soil and exert toxic effects on soil-dwelling microorganisms that play a key role in energy flow and organic matter cycling.[2,3] Heavy metals exert an adverse effect on soil processes. They disrupt microbial metabolic processes associated with macronutrient cycling in the environment, including nitrogen, carbon, phosphorus and sulfur.[4] Specific correlations exist between microorganisms and heavy metals in soil. Heavy metals are bound by microorganisms on the cell surface, which enables them to penetrate microbial cells. The substances secreted by microorganisms form complexes with trace elements and decrease their mobility. Their biodegradation can lead to repeated release of heavy metals, in this case zinc, into the environment.[5] Enzymatic activity is a robust indicator of soil health because it reliably reflects changes in organic matter cycling resulting from contamination including with heavy metals.[6–8] Dehydrogenases are a group of enzymes that are most sensitive to
CONTACT Jadwiga Wyszkowska Olsztyn 10-727, Poland. © 2016 Taylor & Francis Group, LLC
[email protected]
soil contamination with zinc. Those intracellular enzymes are bound to microbial cells, and they are responsible for oxidation–reduction reactions involving organic matter.[9,10] Catalase is also mainly an intracellular enzyme, but it is a less effective indicator of soil fertility than dehydrogenases. The activity of extracellular catalase is very low in the soil environment, and it accounts for less than 2% of its total activity.[11] A weaker relationship between catalase activity and soil fertility has a logical explanation: unlike dehydrogenases which are present in the cells of both aerobic and anaerobic microorganisms, catalase occurs only in the cells of aerobic microorganisms. Urease, extracellular enzyme responsible for nitrogen cycling in soil, is also relatively sensitive to increasing doses of zinc.[12,13] Environmental pollution with heavy metals has been researched extensively in Poland and other countries, but the influence of individual elements on soil biology remains poorly understood.[2,14] Analyses based on a single factor do not produce reliable results, therefore, numerous parameters need to be examined to evaluate zinc’s impact on soil biology. The aim of this study was to evaluate zinc’s influence on the resistance of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase, and urease. The correlations between microbial counts, enzyme activity versus the physicochemical properties of soil, oat and white mustard yields were determined. The results were processed statistically to
University of Warmia and Mazury in Olsztyn, Department of Microbiology, Plac ºodzki 3
262
J. WYSZKOWSKA ET AL.
evaluate the significance of the changes induced by excessive levels of zinc in the soil environment.
Materials and methods Soil The experiment was conducted in a greenhouse of the University of Warmia and Mazury (UWM) in Olsztyn, Poland. Soil samples were collected from the humus horizon in UWM’s Experimental Station in Tomaszkowo (NE Poland, 53.7161 N, 20.4167 E). They were composed of brown soil with granular composition of sandy loam.[15] Soil parameters are presented in Table 1.
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Experimental design Plastic pots were filled with 3 kg of soil each. The experimental variables were: zinc dose applied to soil at 0, 100, 300, 600, 1,200, 2,400, 4,800 mg of Zn2C kg¡1 of soil in the form of ZnCl2 and species of cultivated plant: oat (Avena sativa L.) and white mustard (Sinapis alba). Before sowing, soil samples were fertilized with a single application of macronutrients and micronutrients at the following rates (pure ingredient per mg kg¡1 soil): N – 100 [CO(NH2)]2, P – 44 [KH2PO4], K – 83 [KH2PO4 C KCl], Mg – 25 [MgSO4 ¢ 7H2O], Cu – 5 [CuSO4 ¢ 5H2O], Mn – 5 [MnCl2 ¢ 4H2O], Mo – 5 [(NH4)6Mo7O24 ¢ 4H2O], B – 0.33 [H3BO3]. After fertilization and the addition of zinc chloride to selected samples, soil was thoroughly mixed and placed in plastic pots. Soil moisture content was adjusted to 50% capillary water capacity with the use of tap water. After 24 h, half of the pots were sown with oat cv. Chwat (Avena sativa L.) at 12 plants per pot, and the other half – with white mustard cv. Rota (Sinapis alba) at 8 plants per pot. Soil moisture content was maintained at 50% capillary water capacity throughout the experiment (50 days).
Microorganisms counts and enzyme activity During the growing season, soil samples were subjected to microbiological analyses on experimental days 25 and 50 to determine the abundance of: organotrophic bacteria on Bunt and Rovira medium,[16] actinomyces – on K€ uster and Williams medium supplemented with antibiotics nystatin and actidione,[17] and fungi – on Martin’s glucose peptone agar.[18] Dehydrogenases (EC 1.1) activity was determined using Len[19] € hard’s method modified by Ohlinger, and the activity of
catalase (EC 1.11.1.6) and urease (EC 3.5.1.5) was evaluated based on the method proposed by Alef and Nannipieri.[20] Microorganisms counts were expressed in colony forming units (cfu) which were determined after 5 days of incubation for fungi and after 7 days for organotrophic bacteria and actinomyces. The analysed microorganisms were incubated at 28 C. All enzymatic assays, excluding catalase, were performed using the Perkin-Elmer Lambda 25 spectrophotometer (Waltham, MA, USA). The activity of the tested enzymes was determined with the use of the following substrates: 2,3,5triphenyltetrazolium chloride (TTC, Stanlab, Poland) for dehydrogenases, hydrogen peroxide (Stanlab, Poland) for catalase, and urea (CO(NH2)2 – Eurochem Poland) for urease. Enzyme activity was expressed in the following units per 1 h and 1 kg of soil DM: dehydrogenases – mmol TPF, catalase – mol O2, urease – mmol N-NH4. The soil resistance index[21] (RS) was calculated based on the counts of organotrophic bacteria (Org), actinomyces (Act) and fungi (Fun), as well as the activity of dehydrogenases (Deh), catalase (Cat) and urease (Ure). The following formula was used to calculate the RS index according to Eq. 1.
RS D 1 ¡
2jD0 j ; C0 C jD0 j
(1)
where: RS – soil resistance index, D0 – difference between control soil (C0) and Zn-contaminated soil. The value of the RS index ranged from –1 to 1. RS of 1 indicates that soil’s resistance was not disrupted (full resistance). The lower the values below 1, the greater the loss of soil resistance (lower resistance). Physicochemical properties of soil The granulometric composition of soil was determined before the experiment with the Mastersizer 2000 laser particle size analyzer (Malvern, UK).[22] After harvest, soil pH was determined potentiometrically in an aqueous solution of 1 mol L¡1 KCl, hydrolytic acidity (HAC) and total exchangeable base cations (EBC) were determined by the method proposed by Kappen,[23] total exchangeable cations (TEC), base saturation (BS), and the content of organic carbon (Corg) were determined with the use of the method described by Tiurin,[24] the content of plant-available zinc was determined in accordance with Nelson and Sommers,[24] total zinc content – based on the method
Table 1. Some physicochemical properties of soils used in the experiment. Granulometric composition (mm) 2-0.05
Corg
21
HAC
EBC
TEC
< 0.002
0.05-0.002
(g kg-1)
(%) 72
Ntotal
7
7.05
0.67
Zntotal (mg kg-1)
pHKCl
16.60
7.00
(mmol(C) kg-1 soil) 8.00
111.00
BS % 119.00
93.28
Corg: organic carbon content; Ntotal: total nitrogen content; Zntotal: total zinc content; HAC: hydrolytic acidity; EBC: exchangeable base cations; TEC: total exchangeable cations; BS: base saturation.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
described by PN-ISO 11047,[25] and total nitrogen content – according to PN-EN ISO 20483.[26] Yield of plants The analyzed plants were grown for 50 days. Oat were harvested at BBCH stage 52 – with 20% of inflorescence emerged, and white mustard – at BBCH stage 62 – with 20% of flowers in the first inflorescence open. The above-ground parts of plants were dried at 70 C, they were weighed, and the results were processed statistically.
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Statistical analysis The results were analysed statistically in the Statistica 12.0 software package.[27] Homogeneous groups were determined in Tukey’s test. Differences were regarded as significant at P D 0.05. Zinc’s influence on microbial counts and enzyme activity was determined by Principal Component Analysis (PCA) with multidimensional scaling. The strength of the relationships between variables was determined by calculating linear correlation coefficients. The percentage of variance explained by the analyzed variables was determined by calculating coefficient h2 in ANOVA.
Results and discussion Resistance of microorganisms to soil contamination with zinc The highest doses of zinc disrupted the soil’s biological profile. The results of this study indicate that microbial counts and enzyme activity are influenced by the degree of zinc contamination, species of cultivated plants, and time of exposure (Table 2). Zinc dose was the key determinant of microbial counts. The abundance of organotrophic bacteria, actinomyces and fungi was influenced by Zn2C levels in 80%, 87% and 64%, respectively (Table 2). The resistance of the analyzed microbial groups decreased with a rise in zinc concentrations in soil (Table 3) as demonstrated by the negative values of correlation coefficients between zinc dose and microbial resistance which ranged from –0.678 (for fungi) to –0.916 (for actinomyces). The above results were noted in both oat and white mustard treatments. Organotrophic bacteria and fungi
263
were more resistant in oat than in white mustard pots, whereas the reverse was noted for actinomyces. The analyzed microorganisms were arranged in the following order based on their resistance to zinc (from most to least resistant) to: actinomyces > organotrophic bacteria > fungi. In both oat and white mustard treatments, organotrophic bacteria were more resistant to zinc on day 25 than on to day 50. The reverse was noted in respect of actinomyces whose resistance to soil contamination with zinc increased in both treatments over time. The resistance index of fungi was higher on day 50 than on day 25 in soil sown with oat, whereas the reverse was noted in soil sown with white mustard. The influence of zinc on the analyzed soil microorganisms was determined by Principal Component Analysis (PCA) (Fig. 1). The principal components explained 98.00% of variance in primary variables, where the first principal component explained 78.57%, and the second principal component explained 19.43% of the overall variance. Two homogenous groups were formed around the first principal component. The first group was composed of vectors representing organotrophic bacteria and actinomyces, and the second group comprised fungi. The presence of vectors along the coordinate axes indicates that microbial abundance was influenced by zinc. The abundance of organotrophic bacteria and actinomyces was particularly compromised by higher doses of the analyzed metal (from 1,200 to 4,800 mg kg¡1). Significant negative correlations were observed between zinc dose and the abundance of organotrophic bacteria and actinomyces, whereas fungal counts were positively correlated with zinc concentrations in soil. The counts of organotrophic bacteria and actinomyces were positively correlated with plant yield, enzymes activity and the physicochemical properties of soil, excluding hydrolytic acidity. Fungal abundance was positively correlated only with hydrolytic acidity and the content of plant-available zinc and total zinc (Tables 4 and 5). Microorganisms can differ in their sensitivity to soil contaminants, including zinc. Changes in the structure of microbial communities induced by excessive zinc doses were observed by Moffett et al.[28] and Lock and Janssen.[29] According to the cited authors, microbial groups may develop resistance to selected heavy metals and adapt to the unsupportive conditions in the soil environment. In the present study, the resistance of bacteria, actinomyces and fungi decreased in response to increasing doses of zinc. Zaborowska et al.[30] and
Table 2. Percentage share of observed variability factors h2. Microorganisms Variable factors Dose Zn2C Plant species Soil incubation time Dose Zn2C¢ Plant species Dose Zn2C¢ Soil incubation time Plant species ¢ Soil incubation time Dose Zn2C ¢ Plant species ¢ Soil incubation time
Org: organotrophic bacteria; Act: actinomyces; Fun: fungi. Deh: dehydrogenases; Cat: catalase; Ure: urease.
Enzymes
Org
Act
Fun
Deh
Cat
80.469 0.088 7.030 0.773 9.913 0.588 0.628
86.843 0.202 7.579 0.429 4.018 0.192 0.431
63.918 0.117 18.620 0.423 11.438 0.711 4.240
56.181 4.786 6.909 12.031 12.998 1.265 5.801
77.762 0.234 6.729 2.327 2.659 6.643 3.026
Ure 72.044 2.617 1.116 5.370 16.264 0.001 2.324
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J. WYSZKOWSKA ET AL.
Table 3. Resistance of microorganisms to soil contamination with zinc. Plant species Oat
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Soil incubation time, days Dose Zn2C (mg kg¡1 soil) Organotrophic bacteria 100 300 600 1200 2400 4800 x r Actinomyces 100 300 600 1200 2400 4800 x r Fungi 100 300 600 1200 2400 4800 x r
White mustard
25
50
25
50
0.733a 0.505ab 0.332bc 0.265bc 0.176c 0.138c 0.358 ¡0.766
0.320a 0.284b 0.171c 0.149de 0.125e 0.055f 0.184 ¡0.858
0.381a 0.260b 0.246bc 0.183cd 0.151d 0.136d 0.226 ¡0.774
0.336a 0.228b 0.197b 0.191b 0.088c 0.051c 0.182 ¡0.881
0.176a 0.172ab 0.140b 0.094c 0.084c 0.045d 0.119 ¡0.916
0.436a 0.171b 0.169bc 0.134c 0.046d 0.025d 0.164 ¡0.722
0.270a 0.264a 0.146b 0.113b 0.067c 0.014d 0.146 ¡0.874
0.457a 0.225b 0.222b 0.198b 0.115c 0.067c 0.214 ¡0.772
0.332a ¡0.281b ¡0.283b ¡0.347b ¡0.448bc ¡0.638c ¡0.278 ¡0.741
0.245a ¡0.121b ¡0.607c ¡0.782c ¡0.831c ¡0.835c ¡0.489 ¡0.678
0.576a 0.104ab ¡0.312bc ¡0.310bc ¡0.631c ¡0.723c ¡0.216 ¡0.790
0.706a 0.553a 0.460a ¡0.378b ¡0.426b ¡0.439b 0.079 ¡0.781
Homogeneous groups are denoted with the same letters within microbial groups. r: coefficient of correlation significant at P D 0.05, n D 5
Wyszkowska et al.[2] demonstrated that actinomyces are less sensitive to soil contamination with heavy metals than bacteria. Fungi are also less sensitive to heavy metals than bacteria, and the results of this study confirm this observation. Wyszkowska et al.[31] noted that zinc had a stimulating effect on fungal counts. The heavy metal tolerance of fungi can be partially attributed to their specific defense mechanisms.[32] It is also worth mentioning that mycelia, which are abundantly produced by microscopic fungi, accumulate pollutants, in particular heavy metals.[33] Heavy metals are persistent contaminants in the soil environment, and they are toxic for organisms inhabiting the upmost portion of the lithosphere.[10,34–36] Excessive levels of heavy metals in soil are stressors that compromise microbial biodiversity.[37] The soil resistance index measures the extent to which various stress factors influence the stability and strength of the soil environment.[21] In this study, growing concentrations of zinc had a negative impact on soil biology. The soil resistance index, calculated based on microbial counts and enzyme activity, is a source of valuable information about soil health and soil contamination, including with heavy metals.[8,21]
catalase activity in 78%, and urease activity in 72% (Table 2). The values of the RS index indicate that the resistance of individual enzymes decreased with a rise in zinc dose (Table 6). Those dependencies were confirmed by negative values of correlation coefficients between zinc dose and the SR index which ranged from r D –0.100 (for catalase) to r D –0.867 (for dehydrogenases) on day 25, and from r D –0.517 (for dehydrogenases) to r D –0,839 (for urease) on day 50. Regardless of day, enzyme resistance was generally higher in oat treatments than in soil sown with white mustard. The data shown in Fig. 2 indicate that zinc continued to suppress enzyme activity throughout the experiment. The two principal components explained 96.66% of total variance. The first principal component was negatively correlated with catalase, dehydrogenases and urease. The distribution of cases in the four quadrants of the scatter plot indicates that zinc had a negative effect on the activity of soil enzymes. In the present study, a significant negative correlation (Tables 4 and 5) was observed between enzyme activity, fungal counts, hydrolytic acidity versus the content of plant-available zinc and total zinc. Enzyme activity was significantly positively correlated with plant yield, the counts of organotrophic bacteria and actinomyces, and other physicochemical properties of soil. In addition to microorganisms, soil enzymes are also robust indicators of soil contamination with heavy metals.[2,7,38,39] The most important indicator enzymes are dehydrogenases, catalase and urease which actively participate in soil metabolism and are natural catalysts of various soil processes.[2,40,41] In this study, enzyme resistance decreased with increasing doses of zinc. Dehydrogenases and catalase were most sensitive whereas urease was least sensitive to pollution. Dehydrogenases and catalase are intracellular enzymes, which could explain their heightened susceptibility to environmental and anthropogenic stressors.[2,14] Background pollution levels for live cells are determined based on changes in enzyme activity under exposure to soil contamination with heavy metals.[11] Belyaeva et al.[42] also found that zinc exerts adverse effects on the biochemical properties of soil. In their study, a zinc dose of 300 mg Zn2C kg¡1 inhibited the activity of urease and acid phosphatase. The cited authors suggested that changes in urease and invertase activity can be used as indicators of soil contamination with zinc, lead and copper. The presence of such relationships was confirmed by Chaperon and Sauve.[6] In the present study, dehydrogenases and urease activity decreased significantly with a rise in zinc doses (from 100 to 4,800 mg Zn2C kg¡1 of soil DM). The lowest zinc doses had a stimulating effect on the discussed enzymes. Similar results were reported by Hinojosa et al.[12] who noted a decrease in dehydrogenases and urease activity in soil contaminated with zinc and other heavy metals (As2C, Bi2C, Cd2C, Cu2C, Pb2C, Tl2C).
Physicochemical properties of soil contaminated with zinc Enzyme resistance to soil contamination with zinc Soil contamination with zinc also induced changes in enzyme activity. Zinc influenced dehydrogenases activity in 56%,
Zinc not only compromised the microbiological and biochemical properties of soil, but it also deteriorated soil’s physicochemical profile (Table 7).
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JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
265
Figure 1. Microbial count in the soil contaminated with zinc presented using the PCA method. Dose Zn2C mg kg¡1 of soil: 1 – 0 mg; 2 –100 mg; 3 – 300 mg ; 4 – 600 mg; 5–1200 mg; 6 – 2400 mg; 7 – 4800 mg. Soil incubation time: I – 25 days; II – 50 days. Plant species: o – oat; m – white mustard. Soil microorganisms: Org – organotrophic bacteria, Act – actinomyces, Fun – fungi.
Excessive zinc levels contributed to significant soil acidification, as demonstrated by pH and hydrolytic acidity values. Regardless of plant species, zinc doses of 100–2,400 mg Zn2C kg¡1 soil DM, lowered soil pH by 9%, whereas the highest dose of 4,800 mg of Zn2 induced a 15% decrease in pH. The extent of changes in hydrolytic acidity varied subject to plant species.
In comparison with the control treatment, the highest dose of zinc (4,800 mg of Zn2C) increased hydrolytic acidity by 83% in soil sown with oat, and by as much as 158% in soil sown with white mustard. Regardless of the cause, an increase in soil hydrolytic acidity generally leads to a decrease in exchangeable cations bases in soil which was also observed in the present
Table 4. Correlation between variables for oat. Variable factors Dose Yield Org Act Fun Deh Cat Ure HAC EBC pH Corg TEC BS Ntotal Zntotal
Yield
Org
Act
Fun
Deh
Cat
Ure
HAC
EBC
pH
Corg
TEC
BS
Ntotal
Znav
Zntotal
¡0.990 ¡0.939 ¡0.877 0.987 ¡0.978 ¡0.979 ¡0.913 0.839 ¡0.802 ¡0.933 ¡0.977 ¡0.788 ¡0.777 0.641 0.933 0.934 1.000 0.738 0.649 ¡0.950 0.940 0.821 0.965 ¡0.938 0.906 0.901 0.820 0.893 0.894 ¡0.646 ¡0.936 ¡0.941 1.000 0.980 ¡0.882 0.865 0.961 0.726 ¡0.680 0.649 0.894 0.967 0.638 0.620 ¡0.589 ¡0.809 ¡0.808 1.000 ¡0.802 0.793 0.901 0.629 ¡0.606 0.567 0.841 0.919 0.556 0.539 ¡0.495 ¡0.723 ¡0.722 1.000 ¡0.988 ¡0.956 ¡0.947 0.859 ¡0.811 ¡0.911 ¡0.953 ¡0.795 ¡0.787 0.648 0.931 0.935 1.000 0.935 0.964 ¡0.889 0.823 0.879 0.933 0.803 0.802 ¡0.695 ¡0.944 ¡0.948 1.000 0.832 ¡0.717 0.675 0.879 0.996 0.661 0.644 ¡0.595 ¡0.855 ¡0.856 1.000 ¡0.941 0.897 0.839 0.819 0.882 0.884 ¡0.683 ¡0.962 ¡0.965 1.000 ¡0.974 ¡0.860 ¡0.710 ¡0.962 ¡0.972 0.754 0.959 0.961 1.000 0.880 0.659 0.999 0.999 ¡0.678 ¡0.951 ¡0.950 1.000 0.879 0.878 0.862 ¡0.598 ¡0.919 ¡0.918 1.000 0.643 0.628 ¡0.572 ¡0.838 ¡0.839 1.000 0.998 ¡0.657 ¡0.943 ¡0.941 1.000 ¡0.678 ¡0.939 ¡0.938 1.000 0.734 0.739 1.000 1.000
Org: organotrophic bacteria; Act: actinomyces; Fun: fungi; Deh: dehydrogenases; Cat: catalase; Ure: urease; HAC: hydrolytic acidity; EBC: exchangeable base cations; TEC: total exchangeable cations; BS: base saturation; Corg: organic carbon content, Ntotal: total nitrogen content; Znav: available zinc content; Zntotal: total zinc content; r: coefficient of correlation significant at: P D 0.05, n D 6.
266
J. WYSZKOWSKA ET AL.
Table 5. Correlation between variables for white mustard. Variable factors
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Dose Yield Org Act Fun Deh Cat Ure HAC EBC pH Corg TEC BS Ntotal Zntotal
Yield
Org
Act
Fun
Deh
Cat
Ure
HAC
EBC
pH
TEC
Corg
BS
Ntotal
Znav
Zntotal
¡0.881 ¡0.886 ¡0.914 0.964 ¡0.916 ¡0.986 ¡0.900 0.902 ¡0.906 ¡0.932 ¡0.812 ¡0.902 ¡0.811 0.571 0.919 0.883 1.000 0.858 0.881 ¡0.758 0.985 0.880 0.867 ¡0.718 0.655 0.861 0.822 0.640 0.559 ¡0.533 ¡0.694 ¡0.644 0.862 0.977 0.711 0.619 ¡0.565 ¡0.722 ¡0.669 1.000 0.997 ¡0.763 0.848 0.840 0.669 ¡0.748 0.720 1.000 ¡0.795 0.874 0.873 0.711 ¡0.784 0.755 0.889 0.968 0.746 0.657 ¡0.576 ¡0.758 ¡0.707 1.000 ¡0.820 ¡0.957 ¡0.891 0.887 ¡0.922 ¡0.859 ¡0.662 ¡0.924 ¡0.833 0.480 0.933 0.909 1.000 0.903 0.928 ¡0.777 0.735 0.904 0.818 0.724 0.645 ¡0.534 ¡0.780 ¡0.737 1.000 0.904 ¡0.850 0.858 0.870 0.741 0.856 0.746 ¡0.615 ¡0.873 ¡0.834 1.000 ¡0.801 0.808 0.852 0.612 0.805 0.725 ¡0.511 ¡0.860 ¡0.834 1.000 ¡0.976 ¡0.952 ¡0.700 ¡0.967 ¡0.975 0.305 0.967 0.968 1.000 0.912 0.666 0.999 0.975 ¡0.405 ¡0.994 ¡0.993 1.000 0.848 0.900 0.884 ¡0.394 ¡0.924 ¡0.906 1.000 0.656 0.583 ¡0.538 ¡0.675 ¡0.624 1.000 0.970 ¡0.421 ¡0.994 ¡0.993 1.000 ¡0.230 ¡0.960 ¡0.977 1.000 0.436 0.376 1.000 0.996
Org: organotrophic bacteria; Act: actinomyces; Fun: fungi; Deh: dehydrogenases; Cat: catalase; Ure: urease; HAC: hydrolytic acidity; EBC: exchangeable base cations; TEC: total exchangeable cations; BS: base saturation; Corg: organic carbon content; Ntotal: total nitrogen content; Znav: available zinc content; Zntotal: total zinc content; r: coefficient of correlation significant at: P D 0.05, n D 6.
study. Zinc had a clearly negative impact on soil total exchangeable cations bases. The highest zinc dose (4,800 mg of Zn2C) induced a 41%–52% drop in adsorption capacity, depending on plant species. The zinc-induced acidification and decrease in total exchangeable cations bases, led to a drop in base saturation. In pots contaminated with zinc, base saturation was
negatively correlated with the dose of Zn2C at –0.78 (Table 4) and –0.81 (Table 5). The content of organic carbon (Table 7) was determined by plant species and zinc concentrations. In uncontaminated treatments, organic carbon was more abundant in oat treatments (7.83 g C kg¡1 of soil DM) than in pots sown with white
Table 6. Resistance of enzymes to soil contamination with zinc. Plant species Oat Soil incubation time, days Dose Zn2C (mg kg¡1 soil) Dehydrogenases 100 300 600 1200 2400 4800 x r Catalase 100 300 600 1200 2400 4800 x r Urease 100 300 600 1200 2400 4800 x r
White mustard
25
50
25
50
0.957a 0.861b 0.468c 0.457c 0.082d 0.016d 0.474 ¡0.867
0.915a 0.531b 0.437c 0.242d 0.019e 0.005e 0.358 ¡0.806
0.781a 0.700b 0.310c 0.292c 0.019d 0.002d 0.351 ¡0.820
0.899a 0.110b 0.108b 0.105b 0.020c 0.001c 0.207 ¡0.517
0.177b 0.226b 0.190b 0.361a 0.368a 0.145c 0.245 ¡0.100
0.903a 0.641b 0.146c 0.135c 0.104c 0.094c 0.337 ¡0.624
0.797a 0.581b 0.340c 0.145d 0.137d 0.047e 0.341 ¡0.769
0.763a 0.726a 0.297b 0.149c 0.053cd 0.032d 0.337 ¡0.758
0.298c 0.312c 0.381c 0.659b 0.945a 0.283c 0.480 0.109
0.942a 0.632b 0.469c 0.402d 0.079e 0.062e 0.431 ¡0.839
0.145d 0.330c 0.658b 0.818a 0.282c 0.227cd 0.410 ¡0.251
0.609a 0.370b 0.275c 0.256c 0.119d 0.083d 0.285 ¡0.795
Homogeneous groups are denoted with the same letters within soil enzymes. r: coefficient of correlation significant at P D 0.05, n D 5.
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267
Figure 2. Enzyme activity in the soil contaminated with zinc using the PCA method. Dose Zn2C mg kg¡1 of soil: 1 – 0 mg; 2 – 100 mg; 3 – 300 mg ; 4 – 600 mg; 5 – 1200 mg; 6 – 2400 mg; 7 – 4800 mg. Soil incubation time: I – 25 days; II – 50 days. Plant species: o – oat; m – white mustard. Soil enzymes: Deh – dehydrogenases, Cat – catalase, Ure – urease.
Table 7. Impact of soil contamination with zinc on soil physicochemical properties. mmol(C) kg¡1soil Dose Zn2C (mg kg¡1 soil)
Hydrolytic acidity
Exchangeable base cations
pHKCl
Corg g kg¡1
Total exchangeable cations mmol(C) kg¡1 soil
Base saturation %
9.380 9.630 10.000 10.000 10.130 14.000 17.130 11.815
86.000 85.000 83.000 82.330 81.000 69.000 39.330 73.277
7.000 6.720 6.720 6.680 6.480 6.500 6.120 6.537
7.830 7.580 7.420 7.220 7.020 6.930 6.920 7.182
95.380 94.630 93.000 92.330 91.130 83.000 56.460 85.092
90.170 89.830 89.250 89.170 88.890 83.130 69.650 84.987
6.500 7.380 8.880 9.500 10.750 10.630 16.750 10.648
98.000 91.330 91.330 82.000 77.670 66.330 33.670 73.722
7.220 7.080 6.680 6.680 6.680 6.570 6.100 6.632
7.270 7.050 6.960 6.960 7.010 6.920 6.890 6.965
104.500 98.710 100.210 91.500 88.420 76.960 50.420 84.370
93.780 92.530 91.140 89.610 87.840 86.190 66.770 85.680
0.260 0.140 0.340
1.550 0.830 2.200
0.030 0.010 0.040
0.010 0.010 0.020
1.540 0.820 2.180
0.490 0.260 0.690
Oat 0 100 300 600 1200 2400 4800 x White mustard 0 100 300 600 1200 2400 4800 X LSD0.05 a b ab
x: average for dose 100 – 4800 mg Zn2C. LSD0.05 for: a: zinc dose, b: plant species.
268
J. WYSZKOWSKA ET AL.
Table 8. Content of total nitrogen, available zinc and total zinc in soil contaminated with zinc. (mg kg¡1) Total nitrogen (g kg¡1) 2C
Dose Zn 0 100 300 600 1200 2400 4800 x
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(mg kg
¡1
soil)
Available zinc
Total zinc
Oat
White mustard
Oat
White mustard
Oat
White mustard
0.750 0.830 0.710 0.860 0.740 0.920 0.920 0.830
0.690 0.760 0.680 0.900 0.760 0.900 0.790 0.798
22 64 347 762 711 1370 2320 929
53 119 327 660 714 1330 2390 923
33 82 357 790 753 1470 2430 980
177 139 360 693 763 1390 2810 1026
x: average for dose of 100 to 4800 mg Zn2C.
mustard (7.27 g C). Contamination with Zn2C adversely affected Corg concentrations in soil. Total nitrogen content (Table 8) was determined by the degree of contamination with zinc and plant species. Total nitrogen content increased by 11% on average in contaminated pots and by 16% in white mustard treatments. The content of plant-available zinc and total zinc content increased with the dose, as demonstrated by the high values of correlation coefficients which ranged from 0.88 to 0.93 in pots sown with both plants (Tables 4 and 5). According to the literature[43] heavy metals can contribute to soil acidification. Similar observations were made in this study where pH decreased and hydrolytic acidity increased in pots sown with both oat and white mustard. The above parameters were responsible for the total exchangeable cations bases of soil and its base saturation. Changes in soil pH caused by heavy metals determine the bioavailability of other nutrients for plants.[6,44] Zinc increased the total nitrogen content and decreased the organic carbon content of soil. The increase in nitrogen concentrations in contaminated soil probably resulted from lower nitrogen uptake by oat and white mustard. Plant yield and, consequently, nitrogen uptake, decreased with a rise in zinc dose. The zinc-induced decrease in the organic carbon content of soil could be attributed to the destructive impact of hydrogen ions on organic colloids.[45]
Yield of plants The negative correlation between zinc dose and the yield of the above ground parts of oat (–0.99) and white mustard plants (–0.88) indicates that zinc had a toxic impact on both plants (Fig. 3). The values of correlation coefficients suggest that the analyzed species were characterized by different sensitivity to soil contamination with zinc. Symptoms of poisoning, including disrupted water balance (wilting), chlorosis of young leaves and damage to shoot and root apical meristems, were observed in white mustard plants exposed to zinc doses as low as 300 mg Zn2C kg¡1 of soil, and they were intensified in response to higher doses. A zinc dose of 2,400 mg Zn2C kg¡1 had a nearly lethal effect on plants. Oat responded some what differently to the presence of zinc in soil. The above-ground parts of oat plants were not affected by zinc doses of 100–600 mg Zn2C kg¡1 of soil. A reduction in leaf area and a decrease in oat yield (by 42%) were observed only in response to 1,200 mg Zn2C kg¡1 of soil. The highest zinc dose (4,800 mg Zn2C kg¡1 of soil) lowered oat yield by 98%. The microbiological and biochemical activity of soil is indicative of its fertility and plant yield. According to Yang et al.[41] soil fertility determined by its biological activity significantly influences plant yield. In the present study, excessive zinc concentrations in soil stilted the growth and development of plants. Soil contamination with this heavy metal significantly decreased the yield of oat and white mustard. The two highest
Figure 3. Impact of soil contamination with zinc on plant yield, g DM pot¡1. LSD0.05 for: a – zinc dose, b – plant species.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
doses of zinc had nearly lethal effects on both plants. The negative influence of heavy metals on plants is widely documented in the literature.[31,46–48] Heavy metals can accumulate in plant tissues and disrupt metabolic processes. Heavy metal pollution also intensifies lipid peroxidation, which disrupts calcium homeostasis, contributes to leaf chlorosis and inhibits growth. In a study by Moffett et al.[28] a zinc dose of 400 mg Zn2C kg¡1 DM significantly decreased the yield of peas and spring barley.
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Conclusions Numerous attempts are being made to identify new parameters that could be used as indicators of fertility under exposure to various stressor factors. Soil health can be reliably assessed based on the activity of soil enzymes, the abundance of soil-dwelling microorganisms, the physicochemical properties of soil and plant yield. The above factors are analyzed to observe changes in the soil environment under the influence of zinc. The results of this study indicate that excessive concentrations of zinc have an adverse impact on microbial growth and the activity of soil enzymes. The resistance (RS) of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase, and urease decreased with an increase in the degree of soil contamination with zinc Zinc also exerted an adverse influence on the physicochemical properties of soil and plant development. Oat and white mustard yields decreased under exposure to growing-levels of zinc, and the highest zinc doses of 2,400 and 4,800 Zn2C mg kg¡1 were lethal for plants. Zinc dose was significantly positively correlated with fungal counts, hydrolytic acidity and the content of plant-available zinc and total zinc content, and it was negatively correlated with plant yield, the counts of organotrophic bacteria and actinomyces, enzyme activity, and the remaining physicochemical properties of soil.
Funding This work was supported by the National Science Centre (NCN) as part of grant No. NN304 36 90 38.
References [1] Vrscaj, B.; Poggio, L.; AjmoneMarsan, F. A method for soil environmental quality evaluation for management and planning in urban areas. Landscape Urban Plan. 2008, 88, 81–94. [2] Wyszkowska, J.; Borowik, A.; Kucharski, M.; Kucharski, J. Effect of cadmium, copper and zinc on plants, soil microorganisms and soil enzymes. J. Elem. 2013, 18(4), 769–796. [3] Borowik, A.; Wyszkowska, J.; Kucharski, J.; Bacmaga, M.; Tomkiel, M. Pressure exerted by zinc on the nitrification process. J. Elem. 2014, 19(2), 327–338. [4] Knopf, B.; K€onig, H. Biomethylation of heavy metals in soil and terrestrial invertebrates. In Soil Heavy Metals; Sherameti, I., Varma, A., Eds.; Springer: Berlin/Heidelberg. 2010. [5] Ledin, M. Accumulation of metals by microorganisms: processes and importance for soil systems. Earth-Sci. Rev. 2000, 51, 1–31. [6] Chaperon, S.; Sauve, S. Toxicity interaction of metals (Ag, Cu, Hg, Zn) to urease and dehydrogenase activities in soils. Soil Biol. Biochem. 2007, 39, 2329–2338.
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[29] Lock, K.; Janssen, C.R. Influence of soil zinc concentrations on zinc sensitivity and functional diversity of microbial communities. Environ. Pollut. 2005, 136(2), 275–281. [30] Zaborowska, M.; Wyszkowska, J.; Kucharski, J. Microbiological activity of zinc-contaminated soils. J. Elem. 2006, 11(4), 543–557. [31] Wyszkowska, J.; Boros, E.; Kucharski, J. Effect of interactions between nickel and other heavy metals on the soil microbiological properties. Plant Soil Environ. 2007, 53(12), 544–552. [32] Kabata-Pendias, A. Trace Elements in Soils and Plants, 4th ed.; CRC Press: Boca Raton, FL, 2010; 507. [33] Tripathy, S.; Bhattacharyya, P.; Mohapatra, R.; Som, A.; Chowdhury, D. Influence of different fractions of heavy metals on microbial ecophysiological indicators and enzyme activities in century old municipal solid waste amended soil. Ecol. Eng. 2014, 70, 25–34. [34] Donner, E.; Broos, K.; Heemsbergen, D.; Warne, M.S.J.; McLaughlin, M.J.; Hodson, M.E.; Nortcliff, St. Biological and chemical assessments of zinc ageing in field soils. Environ. Pollut. 2010, 158(1), 339–345. [35] Gomez-Rey, M.X.; Cauto-Vazquez, A.; Gonzalez-Prieto, S.J. Nitrogen transformation rates and nutrient availability under conventional plough and conservation tillage. Soil Till. Res. 2012, 124, 144–152. [36] Trevisan, M.; Coppolecchia, D.; Hamon, R.; Puglisi, E. Potential nitrification, nitrate reductase, and b-galactosidase activities as indicators of restoration of ecological functions in a Zn-contaminated soil. Biol. Fertil. Soils 2012, 48, 923–931. [37] Wysocki, R.; Tamas, M.J. How Saccharomyces cerevisiae copes with toxic metals and metalloids. FEMS Microbiol. Rev. 2010, 34, 925– 951. [38] Dai, J.; Becquer, T.; Rouiller, J.H.; Reversat, G.; Bernhard-Reversat, F.; Lavelle, P. Influence of heavy metals on C and N mineralization and
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ISSN: 0360-1234 (Print) 1532-4109 (Online) Journal homepage: http://www.tandfonline.com/loi/lesb20
Implication of zinc excess on soil health Jadwiga Wyszkowska, Edyta Boros-Lajszner, Agata Borowik, Małgorzata Baćmaga, Jan Kucharski & Monika Tomkiel To cite this article: Jadwiga Wyszkowska, Edyta Boros-Lajszner, Agata Borowik, Małgorzata Baćmaga, Jan Kucharski & Monika Tomkiel (2016) Implication of zinc excess on soil health, Journal of Environmental Science and Health, Part B, 51:5, 261-270, DOI: 10.1080/10934529.2015.1128726 To link to this article: http://dx.doi.org/10.1080/10934529.2015.1128726
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Date: 24 February 2016, At: 08:47
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 2016, VOL. 51, NO. 5, 261–270 http://dx.doi.org/10.1080/10934529.2015.1128726
Implication of zinc excess on soil health Jadwiga Wyszkowska, Edyta Boros-Lajszner, Agata Borowik, Ma»gorzata Bacmaga, Jan Kucharski, and Monika Tomkiel
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Department of Microbiology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
ABSTRACT
ARTICLE HISTORY
This study was undertaken to evaluate zinc’s influence on the resistance of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase and urease. The experiment was conducted in a greenhouse of the University of Warmia and Mazury (UWM) in Olsztyn, Poland. Plastic pots were filled with 3 kg of sandy loam with pHKCl – 7.0 each. The experimental variables were: zinc applied to soil at six doses: 100, 300, 600, 1,200, 2,400 and 4,800 mg of Zn2C kg¡1 in the form of ZnCl2 (zinc chloride), and species of plant: oat (Avena sativa L.) cv. Chwat and white mustard (Sinapis alba) cv. Rota. Soil without the addition of zinc served as the control. During the growing season, soil samples were subjected to microbiological analyses on experimental days 25 and 50 to determine the abundance of organotrophic bacteria, actinomyces and fungi, and the activity of dehydrogenases, catalase and urease, which provided a basis for determining the soil resistance index (RS). The physicochemical properties of soil were determined after harvest. The results of this study indicate that excessive concentrations of zinc have an adverse impact on microbial growth and the activity of soil enzymes. The resistance of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase and urease decreased with an increase in the degree of soil contamination with zinc. Dehydrogenases were most sensitive and urease was least sensitive to soil contamination with zinc. Zinc also exerted an adverse influence on the physicochemical properties of soil and plant development. The growth of oat and white mustard plants was almost completely inhibited in response to the highest zinc doses of 2,400 and 4,800 mg Zn2C kg¡1.
Received 22 August 2015 KEYWORDS
Enzymes; microorganisms; resistance index; soil; zinc
Introduction Environmental richness is one of the key determinants of biodiversity. Countless living organisms reside primarily in soil which hosts various biological and biochemical processes and acts as the ideal intermediary between the edaphon and surface vegetation. Rapid changes that contribute to environmental degradation can decrease the species diversity of living organisms.[1] A serious threat is posed by heavy metals which penetrate soil ecosystems, accumulate in soil and exert toxic effects on soil-dwelling microorganisms that play a key role in energy flow and organic matter cycling.[2,3] Heavy metals exert an adverse effect on soil processes. They disrupt microbial metabolic processes associated with macronutrient cycling in the environment, including nitrogen, carbon, phosphorus and sulfur.[4] Specific correlations exist between microorganisms and heavy metals in soil. Heavy metals are bound by microorganisms on the cell surface, which enables them to penetrate microbial cells. The substances secreted by microorganisms form complexes with trace elements and decrease their mobility. Their biodegradation can lead to repeated release of heavy metals, in this case zinc, into the environment.[5] Enzymatic activity is a robust indicator of soil health because it reliably reflects changes in organic matter cycling resulting from contamination including with heavy metals.[6–8] Dehydrogenases are a group of enzymes that are most sensitive to
CONTACT Jadwiga Wyszkowska Olsztyn 10-727, Poland. © 2016 Taylor & Francis Group, LLC
[email protected]
soil contamination with zinc. Those intracellular enzymes are bound to microbial cells, and they are responsible for oxidation–reduction reactions involving organic matter.[9,10] Catalase is also mainly an intracellular enzyme, but it is a less effective indicator of soil fertility than dehydrogenases. The activity of extracellular catalase is very low in the soil environment, and it accounts for less than 2% of its total activity.[11] A weaker relationship between catalase activity and soil fertility has a logical explanation: unlike dehydrogenases which are present in the cells of both aerobic and anaerobic microorganisms, catalase occurs only in the cells of aerobic microorganisms. Urease, extracellular enzyme responsible for nitrogen cycling in soil, is also relatively sensitive to increasing doses of zinc.[12,13] Environmental pollution with heavy metals has been researched extensively in Poland and other countries, but the influence of individual elements on soil biology remains poorly understood.[2,14] Analyses based on a single factor do not produce reliable results, therefore, numerous parameters need to be examined to evaluate zinc’s impact on soil biology. The aim of this study was to evaluate zinc’s influence on the resistance of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase, and urease. The correlations between microbial counts, enzyme activity versus the physicochemical properties of soil, oat and white mustard yields were determined. The results were processed statistically to
University of Warmia and Mazury in Olsztyn, Department of Microbiology, Plac ºodzki 3
262
J. WYSZKOWSKA ET AL.
evaluate the significance of the changes induced by excessive levels of zinc in the soil environment.
Materials and methods Soil The experiment was conducted in a greenhouse of the University of Warmia and Mazury (UWM) in Olsztyn, Poland. Soil samples were collected from the humus horizon in UWM’s Experimental Station in Tomaszkowo (NE Poland, 53.7161 N, 20.4167 E). They were composed of brown soil with granular composition of sandy loam.[15] Soil parameters are presented in Table 1.
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Experimental design Plastic pots were filled with 3 kg of soil each. The experimental variables were: zinc dose applied to soil at 0, 100, 300, 600, 1,200, 2,400, 4,800 mg of Zn2C kg¡1 of soil in the form of ZnCl2 and species of cultivated plant: oat (Avena sativa L.) and white mustard (Sinapis alba). Before sowing, soil samples were fertilized with a single application of macronutrients and micronutrients at the following rates (pure ingredient per mg kg¡1 soil): N – 100 [CO(NH2)]2, P – 44 [KH2PO4], K – 83 [KH2PO4 C KCl], Mg – 25 [MgSO4 ¢ 7H2O], Cu – 5 [CuSO4 ¢ 5H2O], Mn – 5 [MnCl2 ¢ 4H2O], Mo – 5 [(NH4)6Mo7O24 ¢ 4H2O], B – 0.33 [H3BO3]. After fertilization and the addition of zinc chloride to selected samples, soil was thoroughly mixed and placed in plastic pots. Soil moisture content was adjusted to 50% capillary water capacity with the use of tap water. After 24 h, half of the pots were sown with oat cv. Chwat (Avena sativa L.) at 12 plants per pot, and the other half – with white mustard cv. Rota (Sinapis alba) at 8 plants per pot. Soil moisture content was maintained at 50% capillary water capacity throughout the experiment (50 days).
Microorganisms counts and enzyme activity During the growing season, soil samples were subjected to microbiological analyses on experimental days 25 and 50 to determine the abundance of: organotrophic bacteria on Bunt and Rovira medium,[16] actinomyces – on K€ uster and Williams medium supplemented with antibiotics nystatin and actidione,[17] and fungi – on Martin’s glucose peptone agar.[18] Dehydrogenases (EC 1.1) activity was determined using Len[19] € hard’s method modified by Ohlinger, and the activity of
catalase (EC 1.11.1.6) and urease (EC 3.5.1.5) was evaluated based on the method proposed by Alef and Nannipieri.[20] Microorganisms counts were expressed in colony forming units (cfu) which were determined after 5 days of incubation for fungi and after 7 days for organotrophic bacteria and actinomyces. The analysed microorganisms were incubated at 28 C. All enzymatic assays, excluding catalase, were performed using the Perkin-Elmer Lambda 25 spectrophotometer (Waltham, MA, USA). The activity of the tested enzymes was determined with the use of the following substrates: 2,3,5triphenyltetrazolium chloride (TTC, Stanlab, Poland) for dehydrogenases, hydrogen peroxide (Stanlab, Poland) for catalase, and urea (CO(NH2)2 – Eurochem Poland) for urease. Enzyme activity was expressed in the following units per 1 h and 1 kg of soil DM: dehydrogenases – mmol TPF, catalase – mol O2, urease – mmol N-NH4. The soil resistance index[21] (RS) was calculated based on the counts of organotrophic bacteria (Org), actinomyces (Act) and fungi (Fun), as well as the activity of dehydrogenases (Deh), catalase (Cat) and urease (Ure). The following formula was used to calculate the RS index according to Eq. 1.
RS D 1 ¡
2jD0 j ; C0 C jD0 j
(1)
where: RS – soil resistance index, D0 – difference between control soil (C0) and Zn-contaminated soil. The value of the RS index ranged from –1 to 1. RS of 1 indicates that soil’s resistance was not disrupted (full resistance). The lower the values below 1, the greater the loss of soil resistance (lower resistance). Physicochemical properties of soil The granulometric composition of soil was determined before the experiment with the Mastersizer 2000 laser particle size analyzer (Malvern, UK).[22] After harvest, soil pH was determined potentiometrically in an aqueous solution of 1 mol L¡1 KCl, hydrolytic acidity (HAC) and total exchangeable base cations (EBC) were determined by the method proposed by Kappen,[23] total exchangeable cations (TEC), base saturation (BS), and the content of organic carbon (Corg) were determined with the use of the method described by Tiurin,[24] the content of plant-available zinc was determined in accordance with Nelson and Sommers,[24] total zinc content – based on the method
Table 1. Some physicochemical properties of soils used in the experiment. Granulometric composition (mm) 2-0.05
Corg
21
HAC
EBC
TEC
< 0.002
0.05-0.002
(g kg-1)
(%) 72
Ntotal
7
7.05
0.67
Zntotal (mg kg-1)
pHKCl
16.60
7.00
(mmol(C) kg-1 soil) 8.00
111.00
BS % 119.00
93.28
Corg: organic carbon content; Ntotal: total nitrogen content; Zntotal: total zinc content; HAC: hydrolytic acidity; EBC: exchangeable base cations; TEC: total exchangeable cations; BS: base saturation.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
described by PN-ISO 11047,[25] and total nitrogen content – according to PN-EN ISO 20483.[26] Yield of plants The analyzed plants were grown for 50 days. Oat were harvested at BBCH stage 52 – with 20% of inflorescence emerged, and white mustard – at BBCH stage 62 – with 20% of flowers in the first inflorescence open. The above-ground parts of plants were dried at 70 C, they were weighed, and the results were processed statistically.
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Statistical analysis The results were analysed statistically in the Statistica 12.0 software package.[27] Homogeneous groups were determined in Tukey’s test. Differences were regarded as significant at P D 0.05. Zinc’s influence on microbial counts and enzyme activity was determined by Principal Component Analysis (PCA) with multidimensional scaling. The strength of the relationships between variables was determined by calculating linear correlation coefficients. The percentage of variance explained by the analyzed variables was determined by calculating coefficient h2 in ANOVA.
Results and discussion Resistance of microorganisms to soil contamination with zinc The highest doses of zinc disrupted the soil’s biological profile. The results of this study indicate that microbial counts and enzyme activity are influenced by the degree of zinc contamination, species of cultivated plants, and time of exposure (Table 2). Zinc dose was the key determinant of microbial counts. The abundance of organotrophic bacteria, actinomyces and fungi was influenced by Zn2C levels in 80%, 87% and 64%, respectively (Table 2). The resistance of the analyzed microbial groups decreased with a rise in zinc concentrations in soil (Table 3) as demonstrated by the negative values of correlation coefficients between zinc dose and microbial resistance which ranged from –0.678 (for fungi) to –0.916 (for actinomyces). The above results were noted in both oat and white mustard treatments. Organotrophic bacteria and fungi
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were more resistant in oat than in white mustard pots, whereas the reverse was noted for actinomyces. The analyzed microorganisms were arranged in the following order based on their resistance to zinc (from most to least resistant) to: actinomyces > organotrophic bacteria > fungi. In both oat and white mustard treatments, organotrophic bacteria were more resistant to zinc on day 25 than on to day 50. The reverse was noted in respect of actinomyces whose resistance to soil contamination with zinc increased in both treatments over time. The resistance index of fungi was higher on day 50 than on day 25 in soil sown with oat, whereas the reverse was noted in soil sown with white mustard. The influence of zinc on the analyzed soil microorganisms was determined by Principal Component Analysis (PCA) (Fig. 1). The principal components explained 98.00% of variance in primary variables, where the first principal component explained 78.57%, and the second principal component explained 19.43% of the overall variance. Two homogenous groups were formed around the first principal component. The first group was composed of vectors representing organotrophic bacteria and actinomyces, and the second group comprised fungi. The presence of vectors along the coordinate axes indicates that microbial abundance was influenced by zinc. The abundance of organotrophic bacteria and actinomyces was particularly compromised by higher doses of the analyzed metal (from 1,200 to 4,800 mg kg¡1). Significant negative correlations were observed between zinc dose and the abundance of organotrophic bacteria and actinomyces, whereas fungal counts were positively correlated with zinc concentrations in soil. The counts of organotrophic bacteria and actinomyces were positively correlated with plant yield, enzymes activity and the physicochemical properties of soil, excluding hydrolytic acidity. Fungal abundance was positively correlated only with hydrolytic acidity and the content of plant-available zinc and total zinc (Tables 4 and 5). Microorganisms can differ in their sensitivity to soil contaminants, including zinc. Changes in the structure of microbial communities induced by excessive zinc doses were observed by Moffett et al.[28] and Lock and Janssen.[29] According to the cited authors, microbial groups may develop resistance to selected heavy metals and adapt to the unsupportive conditions in the soil environment. In the present study, the resistance of bacteria, actinomyces and fungi decreased in response to increasing doses of zinc. Zaborowska et al.[30] and
Table 2. Percentage share of observed variability factors h2. Microorganisms Variable factors Dose Zn2C Plant species Soil incubation time Dose Zn2C¢ Plant species Dose Zn2C¢ Soil incubation time Plant species ¢ Soil incubation time Dose Zn2C ¢ Plant species ¢ Soil incubation time
Org: organotrophic bacteria; Act: actinomyces; Fun: fungi. Deh: dehydrogenases; Cat: catalase; Ure: urease.
Enzymes
Org
Act
Fun
Deh
Cat
80.469 0.088 7.030 0.773 9.913 0.588 0.628
86.843 0.202 7.579 0.429 4.018 0.192 0.431
63.918 0.117 18.620 0.423 11.438 0.711 4.240
56.181 4.786 6.909 12.031 12.998 1.265 5.801
77.762 0.234 6.729 2.327 2.659 6.643 3.026
Ure 72.044 2.617 1.116 5.370 16.264 0.001 2.324
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Table 3. Resistance of microorganisms to soil contamination with zinc. Plant species Oat
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Soil incubation time, days Dose Zn2C (mg kg¡1 soil) Organotrophic bacteria 100 300 600 1200 2400 4800 x r Actinomyces 100 300 600 1200 2400 4800 x r Fungi 100 300 600 1200 2400 4800 x r
White mustard
25
50
25
50
0.733a 0.505ab 0.332bc 0.265bc 0.176c 0.138c 0.358 ¡0.766
0.320a 0.284b 0.171c 0.149de 0.125e 0.055f 0.184 ¡0.858
0.381a 0.260b 0.246bc 0.183cd 0.151d 0.136d 0.226 ¡0.774
0.336a 0.228b 0.197b 0.191b 0.088c 0.051c 0.182 ¡0.881
0.176a 0.172ab 0.140b 0.094c 0.084c 0.045d 0.119 ¡0.916
0.436a 0.171b 0.169bc 0.134c 0.046d 0.025d 0.164 ¡0.722
0.270a 0.264a 0.146b 0.113b 0.067c 0.014d 0.146 ¡0.874
0.457a 0.225b 0.222b 0.198b 0.115c 0.067c 0.214 ¡0.772
0.332a ¡0.281b ¡0.283b ¡0.347b ¡0.448bc ¡0.638c ¡0.278 ¡0.741
0.245a ¡0.121b ¡0.607c ¡0.782c ¡0.831c ¡0.835c ¡0.489 ¡0.678
0.576a 0.104ab ¡0.312bc ¡0.310bc ¡0.631c ¡0.723c ¡0.216 ¡0.790
0.706a 0.553a 0.460a ¡0.378b ¡0.426b ¡0.439b 0.079 ¡0.781
Homogeneous groups are denoted with the same letters within microbial groups. r: coefficient of correlation significant at P D 0.05, n D 5
Wyszkowska et al.[2] demonstrated that actinomyces are less sensitive to soil contamination with heavy metals than bacteria. Fungi are also less sensitive to heavy metals than bacteria, and the results of this study confirm this observation. Wyszkowska et al.[31] noted that zinc had a stimulating effect on fungal counts. The heavy metal tolerance of fungi can be partially attributed to their specific defense mechanisms.[32] It is also worth mentioning that mycelia, which are abundantly produced by microscopic fungi, accumulate pollutants, in particular heavy metals.[33] Heavy metals are persistent contaminants in the soil environment, and they are toxic for organisms inhabiting the upmost portion of the lithosphere.[10,34–36] Excessive levels of heavy metals in soil are stressors that compromise microbial biodiversity.[37] The soil resistance index measures the extent to which various stress factors influence the stability and strength of the soil environment.[21] In this study, growing concentrations of zinc had a negative impact on soil biology. The soil resistance index, calculated based on microbial counts and enzyme activity, is a source of valuable information about soil health and soil contamination, including with heavy metals.[8,21]
catalase activity in 78%, and urease activity in 72% (Table 2). The values of the RS index indicate that the resistance of individual enzymes decreased with a rise in zinc dose (Table 6). Those dependencies were confirmed by negative values of correlation coefficients between zinc dose and the SR index which ranged from r D –0.100 (for catalase) to r D –0.867 (for dehydrogenases) on day 25, and from r D –0.517 (for dehydrogenases) to r D –0,839 (for urease) on day 50. Regardless of day, enzyme resistance was generally higher in oat treatments than in soil sown with white mustard. The data shown in Fig. 2 indicate that zinc continued to suppress enzyme activity throughout the experiment. The two principal components explained 96.66% of total variance. The first principal component was negatively correlated with catalase, dehydrogenases and urease. The distribution of cases in the four quadrants of the scatter plot indicates that zinc had a negative effect on the activity of soil enzymes. In the present study, a significant negative correlation (Tables 4 and 5) was observed between enzyme activity, fungal counts, hydrolytic acidity versus the content of plant-available zinc and total zinc. Enzyme activity was significantly positively correlated with plant yield, the counts of organotrophic bacteria and actinomyces, and other physicochemical properties of soil. In addition to microorganisms, soil enzymes are also robust indicators of soil contamination with heavy metals.[2,7,38,39] The most important indicator enzymes are dehydrogenases, catalase and urease which actively participate in soil metabolism and are natural catalysts of various soil processes.[2,40,41] In this study, enzyme resistance decreased with increasing doses of zinc. Dehydrogenases and catalase were most sensitive whereas urease was least sensitive to pollution. Dehydrogenases and catalase are intracellular enzymes, which could explain their heightened susceptibility to environmental and anthropogenic stressors.[2,14] Background pollution levels for live cells are determined based on changes in enzyme activity under exposure to soil contamination with heavy metals.[11] Belyaeva et al.[42] also found that zinc exerts adverse effects on the biochemical properties of soil. In their study, a zinc dose of 300 mg Zn2C kg¡1 inhibited the activity of urease and acid phosphatase. The cited authors suggested that changes in urease and invertase activity can be used as indicators of soil contamination with zinc, lead and copper. The presence of such relationships was confirmed by Chaperon and Sauve.[6] In the present study, dehydrogenases and urease activity decreased significantly with a rise in zinc doses (from 100 to 4,800 mg Zn2C kg¡1 of soil DM). The lowest zinc doses had a stimulating effect on the discussed enzymes. Similar results were reported by Hinojosa et al.[12] who noted a decrease in dehydrogenases and urease activity in soil contaminated with zinc and other heavy metals (As2C, Bi2C, Cd2C, Cu2C, Pb2C, Tl2C).
Physicochemical properties of soil contaminated with zinc Enzyme resistance to soil contamination with zinc Soil contamination with zinc also induced changes in enzyme activity. Zinc influenced dehydrogenases activity in 56%,
Zinc not only compromised the microbiological and biochemical properties of soil, but it also deteriorated soil’s physicochemical profile (Table 7).
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265
Figure 1. Microbial count in the soil contaminated with zinc presented using the PCA method. Dose Zn2C mg kg¡1 of soil: 1 – 0 mg; 2 –100 mg; 3 – 300 mg ; 4 – 600 mg; 5–1200 mg; 6 – 2400 mg; 7 – 4800 mg. Soil incubation time: I – 25 days; II – 50 days. Plant species: o – oat; m – white mustard. Soil microorganisms: Org – organotrophic bacteria, Act – actinomyces, Fun – fungi.
Excessive zinc levels contributed to significant soil acidification, as demonstrated by pH and hydrolytic acidity values. Regardless of plant species, zinc doses of 100–2,400 mg Zn2C kg¡1 soil DM, lowered soil pH by 9%, whereas the highest dose of 4,800 mg of Zn2 induced a 15% decrease in pH. The extent of changes in hydrolytic acidity varied subject to plant species.
In comparison with the control treatment, the highest dose of zinc (4,800 mg of Zn2C) increased hydrolytic acidity by 83% in soil sown with oat, and by as much as 158% in soil sown with white mustard. Regardless of the cause, an increase in soil hydrolytic acidity generally leads to a decrease in exchangeable cations bases in soil which was also observed in the present
Table 4. Correlation between variables for oat. Variable factors Dose Yield Org Act Fun Deh Cat Ure HAC EBC pH Corg TEC BS Ntotal Zntotal
Yield
Org
Act
Fun
Deh
Cat
Ure
HAC
EBC
pH
Corg
TEC
BS
Ntotal
Znav
Zntotal
¡0.990 ¡0.939 ¡0.877 0.987 ¡0.978 ¡0.979 ¡0.913 0.839 ¡0.802 ¡0.933 ¡0.977 ¡0.788 ¡0.777 0.641 0.933 0.934 1.000 0.738 0.649 ¡0.950 0.940 0.821 0.965 ¡0.938 0.906 0.901 0.820 0.893 0.894 ¡0.646 ¡0.936 ¡0.941 1.000 0.980 ¡0.882 0.865 0.961 0.726 ¡0.680 0.649 0.894 0.967 0.638 0.620 ¡0.589 ¡0.809 ¡0.808 1.000 ¡0.802 0.793 0.901 0.629 ¡0.606 0.567 0.841 0.919 0.556 0.539 ¡0.495 ¡0.723 ¡0.722 1.000 ¡0.988 ¡0.956 ¡0.947 0.859 ¡0.811 ¡0.911 ¡0.953 ¡0.795 ¡0.787 0.648 0.931 0.935 1.000 0.935 0.964 ¡0.889 0.823 0.879 0.933 0.803 0.802 ¡0.695 ¡0.944 ¡0.948 1.000 0.832 ¡0.717 0.675 0.879 0.996 0.661 0.644 ¡0.595 ¡0.855 ¡0.856 1.000 ¡0.941 0.897 0.839 0.819 0.882 0.884 ¡0.683 ¡0.962 ¡0.965 1.000 ¡0.974 ¡0.860 ¡0.710 ¡0.962 ¡0.972 0.754 0.959 0.961 1.000 0.880 0.659 0.999 0.999 ¡0.678 ¡0.951 ¡0.950 1.000 0.879 0.878 0.862 ¡0.598 ¡0.919 ¡0.918 1.000 0.643 0.628 ¡0.572 ¡0.838 ¡0.839 1.000 0.998 ¡0.657 ¡0.943 ¡0.941 1.000 ¡0.678 ¡0.939 ¡0.938 1.000 0.734 0.739 1.000 1.000
Org: organotrophic bacteria; Act: actinomyces; Fun: fungi; Deh: dehydrogenases; Cat: catalase; Ure: urease; HAC: hydrolytic acidity; EBC: exchangeable base cations; TEC: total exchangeable cations; BS: base saturation; Corg: organic carbon content, Ntotal: total nitrogen content; Znav: available zinc content; Zntotal: total zinc content; r: coefficient of correlation significant at: P D 0.05, n D 6.
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J. WYSZKOWSKA ET AL.
Table 5. Correlation between variables for white mustard. Variable factors
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Dose Yield Org Act Fun Deh Cat Ure HAC EBC pH Corg TEC BS Ntotal Zntotal
Yield
Org
Act
Fun
Deh
Cat
Ure
HAC
EBC
pH
TEC
Corg
BS
Ntotal
Znav
Zntotal
¡0.881 ¡0.886 ¡0.914 0.964 ¡0.916 ¡0.986 ¡0.900 0.902 ¡0.906 ¡0.932 ¡0.812 ¡0.902 ¡0.811 0.571 0.919 0.883 1.000 0.858 0.881 ¡0.758 0.985 0.880 0.867 ¡0.718 0.655 0.861 0.822 0.640 0.559 ¡0.533 ¡0.694 ¡0.644 0.862 0.977 0.711 0.619 ¡0.565 ¡0.722 ¡0.669 1.000 0.997 ¡0.763 0.848 0.840 0.669 ¡0.748 0.720 1.000 ¡0.795 0.874 0.873 0.711 ¡0.784 0.755 0.889 0.968 0.746 0.657 ¡0.576 ¡0.758 ¡0.707 1.000 ¡0.820 ¡0.957 ¡0.891 0.887 ¡0.922 ¡0.859 ¡0.662 ¡0.924 ¡0.833 0.480 0.933 0.909 1.000 0.903 0.928 ¡0.777 0.735 0.904 0.818 0.724 0.645 ¡0.534 ¡0.780 ¡0.737 1.000 0.904 ¡0.850 0.858 0.870 0.741 0.856 0.746 ¡0.615 ¡0.873 ¡0.834 1.000 ¡0.801 0.808 0.852 0.612 0.805 0.725 ¡0.511 ¡0.860 ¡0.834 1.000 ¡0.976 ¡0.952 ¡0.700 ¡0.967 ¡0.975 0.305 0.967 0.968 1.000 0.912 0.666 0.999 0.975 ¡0.405 ¡0.994 ¡0.993 1.000 0.848 0.900 0.884 ¡0.394 ¡0.924 ¡0.906 1.000 0.656 0.583 ¡0.538 ¡0.675 ¡0.624 1.000 0.970 ¡0.421 ¡0.994 ¡0.993 1.000 ¡0.230 ¡0.960 ¡0.977 1.000 0.436 0.376 1.000 0.996
Org: organotrophic bacteria; Act: actinomyces; Fun: fungi; Deh: dehydrogenases; Cat: catalase; Ure: urease; HAC: hydrolytic acidity; EBC: exchangeable base cations; TEC: total exchangeable cations; BS: base saturation; Corg: organic carbon content; Ntotal: total nitrogen content; Znav: available zinc content; Zntotal: total zinc content; r: coefficient of correlation significant at: P D 0.05, n D 6.
study. Zinc had a clearly negative impact on soil total exchangeable cations bases. The highest zinc dose (4,800 mg of Zn2C) induced a 41%–52% drop in adsorption capacity, depending on plant species. The zinc-induced acidification and decrease in total exchangeable cations bases, led to a drop in base saturation. In pots contaminated with zinc, base saturation was
negatively correlated with the dose of Zn2C at –0.78 (Table 4) and –0.81 (Table 5). The content of organic carbon (Table 7) was determined by plant species and zinc concentrations. In uncontaminated treatments, organic carbon was more abundant in oat treatments (7.83 g C kg¡1 of soil DM) than in pots sown with white
Table 6. Resistance of enzymes to soil contamination with zinc. Plant species Oat Soil incubation time, days Dose Zn2C (mg kg¡1 soil) Dehydrogenases 100 300 600 1200 2400 4800 x r Catalase 100 300 600 1200 2400 4800 x r Urease 100 300 600 1200 2400 4800 x r
White mustard
25
50
25
50
0.957a 0.861b 0.468c 0.457c 0.082d 0.016d 0.474 ¡0.867
0.915a 0.531b 0.437c 0.242d 0.019e 0.005e 0.358 ¡0.806
0.781a 0.700b 0.310c 0.292c 0.019d 0.002d 0.351 ¡0.820
0.899a 0.110b 0.108b 0.105b 0.020c 0.001c 0.207 ¡0.517
0.177b 0.226b 0.190b 0.361a 0.368a 0.145c 0.245 ¡0.100
0.903a 0.641b 0.146c 0.135c 0.104c 0.094c 0.337 ¡0.624
0.797a 0.581b 0.340c 0.145d 0.137d 0.047e 0.341 ¡0.769
0.763a 0.726a 0.297b 0.149c 0.053cd 0.032d 0.337 ¡0.758
0.298c 0.312c 0.381c 0.659b 0.945a 0.283c 0.480 0.109
0.942a 0.632b 0.469c 0.402d 0.079e 0.062e 0.431 ¡0.839
0.145d 0.330c 0.658b 0.818a 0.282c 0.227cd 0.410 ¡0.251
0.609a 0.370b 0.275c 0.256c 0.119d 0.083d 0.285 ¡0.795
Homogeneous groups are denoted with the same letters within soil enzymes. r: coefficient of correlation significant at P D 0.05, n D 5.
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Figure 2. Enzyme activity in the soil contaminated with zinc using the PCA method. Dose Zn2C mg kg¡1 of soil: 1 – 0 mg; 2 – 100 mg; 3 – 300 mg ; 4 – 600 mg; 5 – 1200 mg; 6 – 2400 mg; 7 – 4800 mg. Soil incubation time: I – 25 days; II – 50 days. Plant species: o – oat; m – white mustard. Soil enzymes: Deh – dehydrogenases, Cat – catalase, Ure – urease.
Table 7. Impact of soil contamination with zinc on soil physicochemical properties. mmol(C) kg¡1soil Dose Zn2C (mg kg¡1 soil)
Hydrolytic acidity
Exchangeable base cations
pHKCl
Corg g kg¡1
Total exchangeable cations mmol(C) kg¡1 soil
Base saturation %
9.380 9.630 10.000 10.000 10.130 14.000 17.130 11.815
86.000 85.000 83.000 82.330 81.000 69.000 39.330 73.277
7.000 6.720 6.720 6.680 6.480 6.500 6.120 6.537
7.830 7.580 7.420 7.220 7.020 6.930 6.920 7.182
95.380 94.630 93.000 92.330 91.130 83.000 56.460 85.092
90.170 89.830 89.250 89.170 88.890 83.130 69.650 84.987
6.500 7.380 8.880 9.500 10.750 10.630 16.750 10.648
98.000 91.330 91.330 82.000 77.670 66.330 33.670 73.722
7.220 7.080 6.680 6.680 6.680 6.570 6.100 6.632
7.270 7.050 6.960 6.960 7.010 6.920 6.890 6.965
104.500 98.710 100.210 91.500 88.420 76.960 50.420 84.370
93.780 92.530 91.140 89.610 87.840 86.190 66.770 85.680
0.260 0.140 0.340
1.550 0.830 2.200
0.030 0.010 0.040
0.010 0.010 0.020
1.540 0.820 2.180
0.490 0.260 0.690
Oat 0 100 300 600 1200 2400 4800 x White mustard 0 100 300 600 1200 2400 4800 X LSD0.05 a b ab
x: average for dose 100 – 4800 mg Zn2C. LSD0.05 for: a: zinc dose, b: plant species.
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J. WYSZKOWSKA ET AL.
Table 8. Content of total nitrogen, available zinc and total zinc in soil contaminated with zinc. (mg kg¡1) Total nitrogen (g kg¡1) 2C
Dose Zn 0 100 300 600 1200 2400 4800 x
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(mg kg
¡1
soil)
Available zinc
Total zinc
Oat
White mustard
Oat
White mustard
Oat
White mustard
0.750 0.830 0.710 0.860 0.740 0.920 0.920 0.830
0.690 0.760 0.680 0.900 0.760 0.900 0.790 0.798
22 64 347 762 711 1370 2320 929
53 119 327 660 714 1330 2390 923
33 82 357 790 753 1470 2430 980
177 139 360 693 763 1390 2810 1026
x: average for dose of 100 to 4800 mg Zn2C.
mustard (7.27 g C). Contamination with Zn2C adversely affected Corg concentrations in soil. Total nitrogen content (Table 8) was determined by the degree of contamination with zinc and plant species. Total nitrogen content increased by 11% on average in contaminated pots and by 16% in white mustard treatments. The content of plant-available zinc and total zinc content increased with the dose, as demonstrated by the high values of correlation coefficients which ranged from 0.88 to 0.93 in pots sown with both plants (Tables 4 and 5). According to the literature[43] heavy metals can contribute to soil acidification. Similar observations were made in this study where pH decreased and hydrolytic acidity increased in pots sown with both oat and white mustard. The above parameters were responsible for the total exchangeable cations bases of soil and its base saturation. Changes in soil pH caused by heavy metals determine the bioavailability of other nutrients for plants.[6,44] Zinc increased the total nitrogen content and decreased the organic carbon content of soil. The increase in nitrogen concentrations in contaminated soil probably resulted from lower nitrogen uptake by oat and white mustard. Plant yield and, consequently, nitrogen uptake, decreased with a rise in zinc dose. The zinc-induced decrease in the organic carbon content of soil could be attributed to the destructive impact of hydrogen ions on organic colloids.[45]
Yield of plants The negative correlation between zinc dose and the yield of the above ground parts of oat (–0.99) and white mustard plants (–0.88) indicates that zinc had a toxic impact on both plants (Fig. 3). The values of correlation coefficients suggest that the analyzed species were characterized by different sensitivity to soil contamination with zinc. Symptoms of poisoning, including disrupted water balance (wilting), chlorosis of young leaves and damage to shoot and root apical meristems, were observed in white mustard plants exposed to zinc doses as low as 300 mg Zn2C kg¡1 of soil, and they were intensified in response to higher doses. A zinc dose of 2,400 mg Zn2C kg¡1 had a nearly lethal effect on plants. Oat responded some what differently to the presence of zinc in soil. The above-ground parts of oat plants were not affected by zinc doses of 100–600 mg Zn2C kg¡1 of soil. A reduction in leaf area and a decrease in oat yield (by 42%) were observed only in response to 1,200 mg Zn2C kg¡1 of soil. The highest zinc dose (4,800 mg Zn2C kg¡1 of soil) lowered oat yield by 98%. The microbiological and biochemical activity of soil is indicative of its fertility and plant yield. According to Yang et al.[41] soil fertility determined by its biological activity significantly influences plant yield. In the present study, excessive zinc concentrations in soil stilted the growth and development of plants. Soil contamination with this heavy metal significantly decreased the yield of oat and white mustard. The two highest
Figure 3. Impact of soil contamination with zinc on plant yield, g DM pot¡1. LSD0.05 for: a – zinc dose, b – plant species.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
doses of zinc had nearly lethal effects on both plants. The negative influence of heavy metals on plants is widely documented in the literature.[31,46–48] Heavy metals can accumulate in plant tissues and disrupt metabolic processes. Heavy metal pollution also intensifies lipid peroxidation, which disrupts calcium homeostasis, contributes to leaf chlorosis and inhibits growth. In a study by Moffett et al.[28] a zinc dose of 400 mg Zn2C kg¡1 DM significantly decreased the yield of peas and spring barley.
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Conclusions Numerous attempts are being made to identify new parameters that could be used as indicators of fertility under exposure to various stressor factors. Soil health can be reliably assessed based on the activity of soil enzymes, the abundance of soil-dwelling microorganisms, the physicochemical properties of soil and plant yield. The above factors are analyzed to observe changes in the soil environment under the influence of zinc. The results of this study indicate that excessive concentrations of zinc have an adverse impact on microbial growth and the activity of soil enzymes. The resistance (RS) of organotrophic bacteria, actinomyces, fungi, dehydrogenases, catalase, and urease decreased with an increase in the degree of soil contamination with zinc Zinc also exerted an adverse influence on the physicochemical properties of soil and plant development. Oat and white mustard yields decreased under exposure to growing-levels of zinc, and the highest zinc doses of 2,400 and 4,800 Zn2C mg kg¡1 were lethal for plants. Zinc dose was significantly positively correlated with fungal counts, hydrolytic acidity and the content of plant-available zinc and total zinc content, and it was negatively correlated with plant yield, the counts of organotrophic bacteria and actinomyces, enzyme activity, and the remaining physicochemical properties of soil.
Funding This work was supported by the National Science Centre (NCN) as part of grant No. NN304 36 90 38.
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